Construction of diploid zygotes by interallelic complementation of ade6 in Schizosaccharomyces japonicus

The construction of diploid cells eases genetic analysis in haploid genetic systems because diploid cells allow for the characterization of essential genes. Here, we report the construction of diploid cells using ade6 point mutants that suppress each other via interallelic complementation in the fission yeast Schizosaccharomyces japonicus var japonicus (Sz. japonicus). We constructed an ade6-domK mutant in addition to the previously described ade6-domE. Phenotypes of both mutants exhibited adenine auxotrophy and red colonies. The mutations complemented the phenotypes in a mutually dependent manner. Diploid zygotes, in which the two mutations were introduced simultaneously into the same cells, were isolated by selecting for adenine independence. Such diploid cells are apparently larger in size than haploid cells, yet have a similar nuclear/cytoplasmic ratio, and thus the nuclear size control that has been reported in Sz. pombe is also present in Sz. japonicus.     

Isolation and characterization of the plasma membrane biotin transporter from Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is auxotrophic for biotin (vitamin H) and growth depends on biotin uptake over the plasma membrane. Here a biotin transport mutant of Saccharomyces cerevisiae is used to identify the vht1(+) gene encoding the Schizosaccharomyces pombe plasma membrane transport protein for biotin. SpVht1p belongs to the family of allantoate transporters and has only little sequence homology to the S. cerevisiae biotin transporter. Although having dissimilar primary structures, the biotin transporters in Sz. pombe and S. cerevisiae share similar biochemical properties and regulation. Like in S. cerevisiae, biotin uptake in Sz. pombe is a high-affinity process, is optimal at acidic pH values and inhibited by protonophores, indicating that SpVht1p acts as a proton-biotin symporter. Desthiobiotin, the metabolic precursor of biotin, is also imported by SpVht1p. Deletion of vht1(+) abolishes growth on low external concentrations of the vitamin, showing that vht1(+) encodes the only protein that mediates biotin uptake in Sz. pombe. Expression of vht1(+) is maximal at low external biotin concentrations, indicating that Sz. pombe can adjust the rate of biotin uptake to meet the requirement for the vitamin.     

Marker construction and cloning of a cut1-like sequence with ARS activity in the fission yeast Schizosaccharomyces japonicus

The dimorphic fission yeast Schizosaccharomyces japonicus has proved to be an excellent experimental model for the investigation of the eukaryotic cell. Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote. To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as ars when integrated into a plasmid. Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion. The complementation tests and the recombination frequencies determined in octads of spores identified 28 non-allelic groups (genes) of mutations of the auxotrophic and mycelium-negative mutants. Two groups of linked markers were also identified. The cloned fragment, which expresses ars activity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae).     

Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed. The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe

Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

Genome-wide search of Schizosaccharomyces pombe genes causing overexpression-mediated cell cycle defects

Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail.     

Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.     

Tools and resources for Sz. pombe: a report from the 2006 European Fission Yeast Meeting

Schizosaccharomyces pombe has always suffered from a relative paucity of tools and resources, particularly when compared to Saccharomyces cerevisiae. The European Fission Yeast Meeting, held in March 2006, brought together a significant proportion of the Sz. pombe research community, so it was an ideal opportunity to hold a discussion session on the future needs of those working on this model organism. While the session generated a consensus on the most essential requirements, it also demonstrated the frustrations and concerns of those working with Sz. pombe. The community was also briefed regarding the future transition of the current database (Sz. pombe GeneDB) to a fully-fledged Model Organism Database (MOD) to support the needs of both fission yeast and the broader scientific community.     

DNA sequence of the mat2,3 region of Schizosaccharomyces kambucha shares high homology with the corresponding sequence from Sz. pombe

To define conserved sequences for mat1 imprinting and silencing of the mat2,3 region of Schizosaccharomyces pombe, we determined the DNA sequence of the cognate region (mat2,3 region) of another fission yeast, Sz. kambucha, a yeast species isolated from Kambucha tea mix. The entire mat2,3 region shows more than 98% identity between the two species. Sequence similarity is even higher (99.3%) for mating-type cassettes; deduced amino acid sequences of three of the four Mat peptides (Pi, Pc and Mi) are identical between the two species, while the fourth (Mc) has a single amino acid polymorphism. Comparison of the sequence motif of the imprint site essential for mat1 switching shows that mat-P of Sz. kambucha has a sequence identical to the conserved motif present in Sz. pombe. However, this sequence motif of nine bases differs by one base for mat-M of Sz. kambucha. The sequence of the K region shows about 98% identity between the two species, with the cenH region showing 98.3% homology. Thus, the arrangement of the mat2,3 region in both yeasts is conserved and shows 1-2% nucleotide sequence variation throughout the region. The DNA sequence of the mat2,3 region from Sz. kambucha has been submitted to GenBank under Accession No. AY271822.     

Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2

To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.     

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.     

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.     

Posttranslational activation, site-directed mutation and phylogenetic analyses of the lysine biosynthesis enzymes alpha-aminoadipate reductase Lys1p (AAR) and the phosphopantetheinyl transferase Lys7p (PPTase) from Schizosaccharomyces pombe

Alpha-aminoadipate reductase (AAR), the signature enzyme for lysine biosynthesis in fungi, catalyses the conversion of alpha-aminoadipate to alpha-aminoadipate-semiadehyde in the presence of ATP and NADPH. In Saccharomyces cerevisiae and Candida albicans, the LYS2-encoded AAR is posttranslationally activated by CoA and the LYS5-encoded PPTase. The fission yeast Schizosaccharomyces pombe is evolutionarily highly diverged from S. cerevisiae and C. albicans. We report here several unusual activation characteristics of Sz. pombe Lys1p and Lys7p, isofunctional to Lys2p (AAR) and Lys5p (PPTase), respectively. Unlike the Lys2p from S. cerevisiae and C. albicans, the Sz. pombe Lys1p was active when expressed in E. coli and exhibited significant AAR activity without the addition of CoA or the Sz. pombe Lys7p intron free PPTase. Somewhat higher AAR activity was obtained with the addition of CoA and the Sz. pombe Lys7p PPTase. Substitution of G910A, S913T or S913A in the Sz. pombe Lys1p activation domain (IGGHSI) resulted in no AAR activity. Similarly, substitutions of several amino acid residues in the Sz. pombe Lys7p PPTase domain (G79A, R80K and P81A in Core 1; F93W, D94E, F95W and N96D in Core 1a; G124A, V125I and D126E in Core 2; K172R, E173D and K177R in Core 3) also resulted in no activation of Lys1p and no AAR activity. The Sz. pombe Lys1p amino acid sequence showed a high degree of similarity to other fungal Lys2p proteins; however, the Lys7p amino acid sequence showed much less similarity to other bacterial, fungal and animal PPTases representing several phylogenetic groups.     

Schizosaccharomyces pombe Arc3 is a conserved subunit of the Arp2/3 complex required for polarity, actin organization, and endocytosis

We characterized the Schizosaccharomyces pombe arc3 gene, whose product shares sequence homology with that of the budding yeast ARC18 and human ARPC3/p21 subunits of the Arp2/3 complex. Our data showed that Arc3p co-localizes with F-actin patches at the cell ends, but not with F-actin cables or the equatorial actin ring, and binds other subunits of the Arp2/3 complex. Gene deletion analysis showed that arc3 is essential for viability. When arc3 expression was repressed, F-actin patches became dispersed throughout the cell with greatly reduced mobility. Furthermore, in arc3-repressed cells, endocytosis was also inhibited. Human ARPC3 rescued the viability of the Sz. pombe arc3 null mutant; in addition, ARPC3 also localized to F-actin patches in human cells. These data suggest that Arc3p is an evolutionarily conserved subunit of the Arp2/3 complex required for proper F-actin organization and efficient endocytosis.     

Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP-galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP-galactose transporter. We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe, and found that the gms1(+) gene encodes a UDP-galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight-membrane-spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1-GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation-defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP-galactose transport activity but no change in the localization to the Golgi membrane. The C-terminal truncated Gms1p mutants demonstrated that the C-terminal hydrophilic region was dispensable for targeting and function as UDP-galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C-terminus-proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane.